Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.

OBJECTIVES To investigate and characterize Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease, attendants of animals with suspected infection, and healthy humans, using multiple diagnostic tests. METHODS A total of 119 samples (35 stool, 76 serum, three blood clots, and five biopsies) were collected from five patients with Crohn's disease, eight attendants of animals with Johne's disease, and 93 apparently normal control subjects (Agra region) from North India. Samples were screened for the presence of MAP by smear examination, culture of stool, blood clot and biopsies, and ELISA. Colonies obtained by culture were further characterized using polymerase chain reaction (PCR) with IS900 MAP-specific primers. RESULTS Using all diagnostic modalities, MAP and/or MAP antibodies were identified in 100% (5/5) of subjects with Crohn's disease; 75.0% (6/8) of attendants of MAP infected animals were positive and 38.0% (27/71) of apparently normal controls were also positive. Most sensitive test was ELISA (100%, 5/5), followed by culture (80.0%, 4/5), and acid-fast staining. Ziehl-Neelsen staining was positive in 37.5% (3/8) of subjects with active animal husbandry practices. In 71 serum samples from control subjects, seroprevalence of MAP was 38.0% using indigenous protoplasmic antigens (PPA) and 36.6% using commercial PPA. Of the serum samples from the Crohn's disease patients, 100% (5/5) were positive by ELISA using indigenous PPA and 40.0% (2/5) were positive by ELISA using commercial PPA. IS900 PCR was used to characterize tiny colonies of MAP that grew extremely slowly on Herrold's egg yolk medium, and of 15 (42.8%) cultures, 14 (93.3%) were typed as MAP. CONCLUSIONS Paper documented the presence of MAP in all patients with Crohn's disease, in some animal attendants who had the history of working with goat herds infected with Johne's disease and in few normal healthy individuals. Presence of Ziehl Neelsen positive MAP. In the stool of attendants working with MAP-infected animals was unique to humans. ELISA based on antigens derived from indigenous MAP 'bison type' genotype of goat origin was most sensitive modality for screening Crohn's disease patients.